Genome-wide prediction and functional analysis of WOX genes in blueberry

Background WOX genes are a class of plant-specific transcription factors. The WUSCHEL-related homeobox (WOX) family is a member of the homeobox transcription factor superfamily. Previous studies have shown that WOX members play important roles in plant growth and development. However, studies of the WOX gene family in blueberry plants have not been reported. Results In order to understand the biological function of the WOX gene family in blueberries, bioinformatics were used methods to identify WOX gene family members in the blueberry genome, and analyzed the basic physical and chemical properties, gene structure, gene motifs, promoter cis-acting elements, chromosome location, evolutionary relationships, expression pattern of these family members and predicted their functions. Finally, 12 genes containing the WOX domain were identified and found to be distributed on eight chromosomes. Phylogenetic tree analysis showed that the blueberry WOX gene family had three major branches: ancient branch, middle branch, and WUS branch. Blueberry WOX gene family protein sequences differ in amino acid number, molecular weight, isoelectric point and hydrophobicity. Predictive analysis of promoter cis-acting elements showed that the promoters of the VdWOX genes contained abundant light response, hormone, and stress response elements. The VdWOX genes were induced to express in both stems and leaves in response to salt and drought stress. Conclusions Our results provided comprehensive characteristics of the WOX gene family and important clues for further exploration of its role in the growth, development and resistance to various stress in blueberry plants. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10356-5.


Background
The WOX protein belongs to the homeobox (HOX) superfamily [1], and is composed of 65 amino acids, which fold into a DNA-binding domain with three helixes in space [2].Homeodomain is characterized by a highly conserved "helix-loop-helix-turn-helix, with all WOX members containing a homologous domain of 60-66 amino acid residues [3].The sequence specificity of the domain distinguishes the WOX gene family from other homeobox.The homology domain binds to DNA via a helix-turn-helix (HTH) structure.The characteristic of the HTH motif is that two α-helices tightly connected to DNA and linked through a loop [4].
In plants, members of the WOX gene family play important roles in developmental processes, including embryonic development, maintenance of meristematic tissue stem cells, development of lateral organs, seed formation, and regeneration of detached tissues and organs [5].The WOX gene family has been identified in many plants, such as maize [6], apple [7], rice [8] and soybean [5], including 15 WOX genes in Arabidopsis.Based on their evolutionary relationships, they are classified into three branches, the WUS branch (WUS Clade), Intermediate Clade and Ancient Clade [3,4].The members of the blueberry WOX gene family, like those of the Arabidopsis WOX gene family, are distributed among three clades.The WUS Clade promotes stem cell proliferation in floral meristems, the Intermediate Clade demonstrates effective expression during embryonic development and cell division processes, and the Ancient Clade functions in root development and floral transition [5].Several members of the Arabidopsis WOX family have been shown to be important for maintaining embryonic patterns in stem cells, stem apical meristem (SAM), root apical meristem and organs formation [9].The AtWUS proteins are involved in maintaining the homeostasis of stem cells in SAM at all developmental stages, and the homeostasis of SAM is disrupted in Arabidopsis wus mutants [10].Similarly, VdWOX3, belonging to the WUS Clade, exhibits the highest expression level in stems, indicating its potential significant role in stem development.The AtWOX2 protein is expressed in germ cells and is required for embryonic tip development [11].AtWOX3 is expressed in the leaf primordium and floral organ margins, and forms the peripheral histological structures of nutrient and floral organs [12].In rice (Oryza sativa), OsWOX3 is involved in organ development, growth of leaf lateral axes and vascular formation, morphogenesis of spikelet lemma and palea, and development of tillers and lateral roots [13,14].AtWOX4 plays a crucial role in the regulation of growth hormone in the formation layer [15].AtWOX5 is necessary for the maintaining the homeostasis mechanism of IAA in root tip meristem tissues [16].AtWOX6 is a homologous domain protein encoded by the PRETTY FEW SEEDS2 gene, which plays a regulatory role in ovule development [17].AtWOX6 (also known as HOS9-1) plays a role in the physiological processes of plant resistance to freezing by affecting gene activity independent of the CBF pathway [18].AtWOX7 regulates the development of plant lateral roots as well as the distribution of sugars [19].In blueberries, VdWOX9 exhibits higher expression levels in roots compared to other tissues, which may suggest its involvement in regulating root development, given its closer phylogenetic relationship with AtWOX7 in the evolutionary tree.AtWOX8 regulates growth hormone gradient formation during early embryonic development [20].STIMPY (STIP; also known as WOX9) may play a role in organisation by maintaining cell division and preventing premature differentiation, and STIP identifies a new genetic pathway that combines developmental signaling with cell cycle control [21].Moreover in wheat, TaWOX9 promotes root development [22].
In Arabidopsis, WOX11 associates upstream growth hormone signaling with downstream transformation of cell morphology to initiate the formation of root and healing tissue primordia [23].Several studies have found that the switch in expression from WOX11/12 to WOX5/7 is crucial for the formation of root primordia during new root organogenesis in plants [24].WOX11 and WOX12 are involved in the life cycle of the most primitive cells that form root organs in Arabidopsis [25].AtWOX13 is involved in initiation and development of primordial and lateral root, and is dynamically expressed during pistil and embryonic development.The WOX13 promotes replicon development by blocking the activity of JAG/FIL genes in the inner tissues [26,27].In contrast, overexpression of WOX14 stimulates the expression of GA3ox anabolic genes and represses GA2ox catabolic genes, which promotes the accumulation of bioactive GA.WOX14 promotes the differentiation and lignification of vascular cells in the inflorescence stems of Arabidopsis [28].
WOXs gene not only play a role in plant development, but also in response to abiotic stresses.AtWOX6 (also known as HOS9-1) plays a role in the physiological processes of plant resistance to freezing by affecting gene activity independent of the CBF pathway [18].Mutant alleles of AtWOX6 are expressed in response to freezing stress [18].In rice, most WOXs gene respond to abiotic stress stimuli such as drought, salt and cold [8].In blueberries, after salt treatment, the expression levels of VdWOX4 and VdWOX11 are upregulated.Following drought treatment, the expression levels of VdWOX4 and VdWOX8 are upregulated, while those of VdWOX7 and VdWOX9 are downregulated.These phenomena collectively suggest the response of the blueberry WOX family to abiotic stressors.In woody plants grapes, the VvWOX genes is a key regulator of somatic embryogenesis in grapes [29].In the gymnosperm European spruce, it contains a number of WOX related genes, many of which are expressed during embryonic development [30].Interestingly, this study revealed through phylogenetic tree and collinearity analysis that blueberries share a closer evolutionary relationship with grapes, both being woody plants.Gene structure analysis further revealed that the number of introns ranges from 1 to 3. Similar results were also observed in leguminous plant species.
In summary, WOX transcription factors mainly affect plant growth and development by altering the expression of downstream genes.It is noteworthy that the cloning and functional studies of WOX proteins mainly focus on model plants such as Arabidopsis and rice.However, no comprehensive molecular evolutionary studies have been conducted on blueberry (evergreen blueberry: Vaccinium darrowii).Blueberry (Vaccinium darrowii) is one of the top five health foods for human beings and is recognised as the "King of Fruits of the World", and given the current status of blueberries as a bioactive ingredient and valuable food, they have greater nutritional and health potential [31].
In this study, we comprehensively identified and analyzed the structure, motifs, cis elements and homology characteristics of the WOX gene family in blueberry.Twelve WOX genes were identified from the entire genome of sequenced evergreen blueberry, and their functions were predicted through phylogenetic analysis and quantitative analysis of different tissues, This study will provide further insights into the evolution and functional characteristics of the WOX gene family in blueberry and also provides evidence for further research on the role of the WOX family in growth, development and stress resistance.To provide genetic resources and scientific basis for breeding high-yielding, high-quality, and stress-resistant blueberry cultivars.

Identification of blueberry VdWOX gene family members
To identify the WOX family in the blueberry genome, we employed two distinct methodologies.Firstly, The nucleotide and protein sequences of blueberry were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/), and the WOX family protein homeodomain feature file was downloaded from the website Pfam (https://pfam.xfam.org/)(PF00046), built a Hidden Markov Model using HMMER 3.0, and searched for proteins containing homeodomain in the above mentioned database (NCBI) using the hmm search programme in HMMER 3.0, set the E-value threshold to 0.01, and redundant protein sequences were manually removed.Secondly, a local BLASTp was conducted against the blueberry genome database using 15 known Arabidopsis sequences to search for WOX protein sequences, with an E-value threshold set at 1 × 10 − 5.A summary of the results from both methods was provided, and redundant sequences were eliminated, resulting in a total of 15 protein sequences identified as candidate WOX family proteins in blueberries.Their conserved domains were validated using NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd/).The ExPASy online website (https://web.expasy.org/protparam/)was used to predict the physicochemical properties of the VdWOX gene family, including the number of amino acids, molecular weight, isoelectric point, hydrophilicity and the subcellular localization of VdWOXprotein was predicted using the website (http:// www.csbio.sjtu.end.cn/bioinf/Cell-PLoc-2/).
Chromosome location, sequence alignment, gene structure, conserved motifs and three-dimensional domain analysis.
The chromosomal location and gene structure of VdWOX genes were visualized using TB tools based on information from the GFF annotation file (NCBI) [32].WOX protein sequence comparison was performed using DNAMAN.Conserved motifs were analyzed using the online website MEME (https://meme-suite.org/meme/).Perform secondary structure analysis of blueberry WOX gene family proteins using SOPMA (https://npsa-prabi. ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html).The VdWOX protein sequence were analyzed for three dimensional domain using the online tool SWISS-MODEL (https://swissmodel.expasy.org/).To ensure the accuracy of the model, AtWOX protein with high similarity to VdWOX protein were used as templates.

Evolutionary tree analysis of different species of WOXs
To construct the phylogenetic tree of WOXs, the protein sequences of WOXs from Arabidopsis thaliana [33] and maize [34] were extracted from the previous study, and the protein sequences of WOXs from grape and barley were downloaded from the PlantTFDB (http://planttfdb.gao-lab.org/)database.Phylogenetic trees were constructed by the method of nearest-neighbour joining (NJ) after multiple sequence comparison using MEGA X software with a self-expansion value of 1000 [35].The relevant species protein sequences are listed in Table S1.

Synteny relationship analysis
Blueberry genome annotation files (GFF files) were downloaded from the NCBI database.The Multiple Covariance Scanning Toolkit from TBtools was used to visualize the covariance relationships of WOXs from different species.

Identification of cis-acting elements in VdWOX promoter
The 2 kb upstream of each VdWOX genes was obtained from the NCBI database as promoter region.The cisacting elements were predicted on Plant-CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and analyzed using Excel for classification [36].

Plant materials, growth conditions and stress treatments
In this study, O'Neill blueberry seedlings were used as the experimental material.One year old blueberry seedlings were grown in a plant growth chamber with a photoperiod of 16 h of light and 8 h of darkness, a light intensity of 100 µmolm -2 s -1 at 23℃ (light) and 20℃ (dark).Different tissue parts such as roots, stems, leaves, flowers, young fruits as well as mature fruits of normal growing blueberries were taken.

RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using FastPure Universal Plant Total RNA Isolation Kit and cDNA strands were synthesised using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme).VdTub2 was used as a control, and quantitative PCR was performed with a CFX Connect Real-Time System (Bio-Rad, America) using the ChamQ Universal SYBR qPCR Master Mix (Vazyme).VdWOX genes specific primers were designed using Premier 5.0 software, and the sequences are listed in Table S2.Reaction conditions: pre-denaturation: 1 min at 95 °C, denaturation: 15 S at 95 °C, annealing extension: 15 S at 60 °C and cycling 40 times.The method of comparing CT values was used to assess the relative expression level of qRT-PCR products [37].

Statistical analysis
All data were analyzed with Origin 8 and SPSS19.Twotailed Student's t test was used for statistical analysis.

Identification of blueberry WOX gene family members
To understand the possible biological functions of WOX protein in blueberries, we searched the hidden Markov model (HMM) of homeodomain.After the selection of conserved domains in the potential candidate proteins, a total number of 12 WOX encoding genes were identified in the blueberries genome.Based on their chromosomal locations, they were designated as VdWOX1-VdWOX12, respectively (Table 1).The amino acid numbers of various proteins encoded in the blueberry WOX gene family range from 171 to 447.The molecular weight range was 19839.3(VdWOX9) − 48547.2(VdWOX4).The isoelectric point was within the range of 5.46 (VdWOX6) − 9.92 (VdWOX11).The instability index range was 44.85 (VdWOX5) − 68.93 (VdWOX12), and the average hydrophilicity coefficients were all negative, indicating that they were all hydrophilic proteins.Subcellular localization prediction indicates that all VdWOX proteins are localized in the nucleus.

Chromosomal localization, developmental tree and multiple sequence comparison analysis of VdWOX gene family
According to genetic mapping information, the 12 WOX gene family members in blueberries were located on 8 chromosomes (Fig. 1).Among them, there were no WOX gene family members on chromosomes 3, 4, 8 and 9.There was only one member of the WOX gene family on chromosomes 1, 2, 5, 6, 10 and 11.And chromosomes 7 and 12 each had three members of the WOX gene family.
In order to better reveal the functional domains in the VdWOX gene family, we performed a developmental tree analysis of 12 VdWOX proteins sequences (Fig. 2A), and classified the VdWOX family proteins into three subfamilies (WUS Clade, Ancient Clade and Intermediate Clade).Meanwhile, we performed a simple modular structure study of the VdWOX proteins sequences using (SMART) (Fig. 2B) and found that all the proteins contained the structural feature of homeodomain, while the members of the WUS Clade subfamily contained the WUS-box motif, highlighting the defining characteristics of the VdWOX gene family.
Sequence comparison showed that the distribution of amino acids in the conserved homeodomain of the 12 VdWOX proteins was extremely similar (Fig. 2C).Previous studies reported the presence of 11 conserved amino acids in the homeodomain region, including Q, L and Y in Helix 1 and I, V, W, F, N, K and R in Helix 3 [31].These amino acids are also conserved in the homeodomain of VdWOX proteins.The conserved amino acid residues in Helix 2 were also found to be P, I and L, as well as F and Q in Helix 3. Proteins in the WUS Clade subfamily were found to contain WUS-box motifs with the sequence "TLXLFP" (where X represents any amino acid) (Fig. 2D).
Simultaneously.The secondary structure of WOX family proteins in blueberry was analyzed by SOPMA.The results indicate that the secondary structure of these 12 blueberry WOX family proteins comprise four structural forms: Alpha helix, Extended strand, Beta turn and Random coil.Among these, Random coil constitutes the majority, exceeding 47% in all cases, while Beta turn is the least abundant, ranging between 3% and 6% (Fig. 2E).

Phylogenetic analysis of the WOX gene family
To investigate the phylogenetic relationship of WOX genes, a total of 82 WOX protein sequences from Arabidopsis, maize (Zea mays), grape (Vitis vinifera), barley (Hordeum vulgare) and blueberry (Vaccinium darrowii) were used for constructing a phylogenetic tree (Fig. 3).A phylogenetic tree was constructed for the WOX family of five species.In addition, the homology relationships among the WOX gene family of Arabidopsis, maize, grape, barley and blueberry were also investigated (Fig. 3).It was found that members of maize, barley and Arabidopsis as herbaceous plants appeared to cluster with each other, such as AtWOX11 and AtWOX12 in Arabidopsis at the Intermediate Clade, HvWOX7 and HvWOX8 in barley, ZmWOX21 and ZmWOX23 in maize.Similarly, clustering phenomena have also been observed in Nelumbo nucifera, such as NnWOX6 and NnWOX9, NnWOX1 and NnWOX4 [38].Previous studies have found that the relationship between GmWOXs and PvWOXs is closer, possibly due to the fact that lotus root and soybean both belong to the legume family [5].The same phenomenon occurred with members of grapes and blueberries as woody plants, such as VdWOX6 and VvWOX4 in the ancient Clade and VdWOX5 and VvWOX2 in the WUS Clade.This indicated that blueberry and grape were closely related as woody plants.The protein IDs of the required species are shown in Table S1.

Gene structure and motif analysis of WOX gene family in blueberry
To investigate the structural diversity of VdWOX genes, the distribution of exons and introns was analyzed.Gene structure analysis revealed that the number of introns among members of the VdWOX gene family ranges from 1 to 3, a phenomenon also observed in lotus and soybean [5,38].Specifically, SVdWOX9, VdWOX10, VdWOX11 and VdWOX12 contain two exons, VdWOX2 and VdWOX8 contain four exons, while the remaining six members contain three exons each (Fig. 4).Moreover, to reveal the correlations of WOX gene family, their conservative motifs were predicted.Conservative motifs analysis showed that all VdWOX family proteins contained motif 1 and motif 2, and motif 1 appeared before motif 2 in all cases.In the WUS Clade subfamily, WOX proteins all contain motif 3, and in the Ancient Clade subfamily VdWOX6 contains only motif 1 and motif 2.Moreover, in the Intermediate Clade subfamily both VdWOX1 and VdWOX4 contain motif 6.Among the 10 motifs, Motif 1 and Motif 10 comprise 50 amino acids each, whereas Motif 8 and Motif 9 contain 7 and 8 amino acids respectively (Fig. 4C).To some extent, the specificity of the motifs in different subfamilies may lead to functional differences in the VdWOX genes.In addition, two pairs of genes (VdWOX3 and VdWOX5, VdWOX2 and VdWOX8) had very high similarity in gene structure and conserved motifs, which indicated that they might have redundant functions in blueberry.

Three-dimensional structure analysis of VdWOX gene family
In order to understand the structure of VdWOX proteins, a VdWOX proteins model was constructed using AtWOX proteins as a template on SWISS-MODEL.The results showed that although the 12 VdWOX proteins differed in length and morphology, they all contained a conserved homeodomain, which is typical WOX proteins in plants (Fig. 5).The homeodomain was characterised as helix-loop-helix, which was composed of 60-66 amino acid residues.The same observation was obtained from the sequence comparison analysis (Fig. 2C).

Synteny analysis of the WOX gene family
Gene duplication occurs universally during plant evolution, including whole genome duplication, tandem duplication, segmental duplication and gene duplication can produce homologous genes that share sequence similarities.Synteny analysis was performed to determine the evolutionary characteristics of the WOX gene family.Intraspecies collinearity analysis of VdWOX gene family and interspecies collinearity analysis with Arabidopsis revealed two pairs of fragment duplication events in members of the VdWOX family of genes: VdWOX2 and VdWOX8, VdWOX3 and VdWOX5 (Fig. 6A).Whereas 10 pairs of genes had collinearity in the VdWOXs and AtWOXs: VdWOX2 and AtWOX6, VdWOX4 and AtWOX8, VdWOX6 and AtWOX13, VdWOX7 and AtWUS1, VdWOX7 and AtWOX14, VdWOX8 and AtWOX6, VdWOX9 and AtWOX5, VdWOX9 and AtWOX7, VdWOX10 and AtWOX3, VdWOX10 and AtWOX2.In the phylogenetic analysis, VdWOX7 and AtWUS1 were in the same branch (Fig. 3), which was consistent with the results of the covariance analysis, suggesting that VdWOX7 and AtWUS1 may have similar functions in plant growth and development as well as in stress response.Simultaneously, rice and grape were also selected for collinearity analysis with blueberry.The results revealed that in the comparison between blueberry and japonica rice, 4 pairs of genes exhibited collinear relationships (Fig. 6B).Similarly, in the comparison between blueberry and grape, 11 pairs of genes were found to have collinear relationships, indicating a high degree of homology between the VdWOX genes of blueberry and the VvWOX genes of grape, both being woody plants (Fig. 6B), suggesting a close relationship between them.Furthermore, the results indicated that in the collinearity with the genomes of monocotyledonous and dicotyledonous plants, blueberry exhibited one-to-many or many-toone orthologous genes.These genes underwent multiple duplication events, implying intimate phylogenetic relationships among species.Their evolutionary functions are likely conserved, with their ancestral functions remaining unchanged or unaltered during duplication, thus playing crucial roles in the evolution of the WOX gene family.

VdWOXs promoter regions contain the key cis-elements for phytohormone and stress response
Cis-elements are non-coding DNA sequences in the promoter region of a gene, which are crucial for gene expression and widely involved in the regulation of plant growth, development, and stress response [39].To better understand the regulatory network of VdWOX genes, we analyzed their promoter regions.Further analysis of the promoter regions of the VdWOX gene family revealed that they all contain cis-elements associated with plant growth and development, phytohormones and stress responses (Fig. 7A).The response elements identified in the 2000 bp promoter region upstream of the blueberry WOX family genes can be classified into three categories based on their functions.Of these, 29 elements were related to plant growth and development, 72 elements were related to phytohormone response pathways and 175 elements were related to abiotic and biotic stresses (Fig. 7B).The most abundant cis-elements in the promoter region of VdWOX family genes were salt stress response element (MYC), stress response element (STRE), anaerobic stress-related element (ARE), abscisic acid response element (ABRE) and growth hormone response element (AAGAA-motif ), suggesting that the VdWOX gene family may be involved in a variety of pathways of growth, development and stress response in blueberry.

Analysis of VdWOX gene family expression patterns
In Arabidopsis, AtWOX genes were abundant in roots, leaves, stems, flowers and seeds [40].In rice OsWOX were involved in the development of all organs [34].In paper mulberry, most of the BpWOX had relatively high expression in terminal yards and stems, and BpWOX1 was mainly expressed in terminal buds and leaves [41].In Citrus sinensis, CsWUS was most highly expressed in flowers and stems, while CsWOX1 and CsWOX2 were constitutively expressed in all tissues [42].In this study, we found high expression of VdWOX genes in stems, leaves and mature fruits (Fig. 8), which suggested that they played an important role in blueberry plant growth increase and fruit development.In particular, VdWOX2 had the highest expression in leaves, indicating that VdWOX2 might be involved in leaf formation and physiological processes.

VdWOX genes expression in response to salt and drought stress in blueberry
Previous studies had shown that WOX genes played an important role in regulating plant responses to abiotic stresses [8].In rice, drought tolerance was enhanced in transgenic overexpressing OsWOX13 [43].In poplar, PagWOX11/12a responsed to drought stress by promoting root elongation and biomass increase, thereby enhancing drought tolerance in poplar [44,45].In apple, MdWOX13-1 improved drought tolerance by enhancing the ability to scavenge reactive oxygen species [46].
To investigate the response of VdWOX genes to abiotic stresses, blueberry seedlings were treated with 200 mM NaCl, and samples were taken at 0 d, 1 d, 3 d, 5 d, 7 d and 9 d respectively.Quantitative PCR analysis showed that the expression of all VdWOX genes was responsive to salt stress (Fig. 9).The expression of 5 genes (VdWOX1, VdWOX4, VdWOX7, VdWOX9 and VdWOX11) was up-regulated after treatment.The expression of 5 genes (VdWOX2, VdWOX3, VdWOX6, VdWOX8 and VdWOX10) was down-regulated and then up-regulated.And the expression of 2 genes (VdWOX5 and VdWOX12) was down-regulated.Moreover, the expression of 6 genes (VdWOX1, VdWOX2, VdWOX3, VdWOX6, VdWOX10 and VdWOX11) reached the highest level at 9 d after treatment.
Regarding drought treatment, samples were taken at 1 d, 3 d, 6 d, 9 d, 12 d and 15 d of water deficit.Quantitative PCR analysis showed that the expression of all VdWOX genes appeared differentially in response to drought stress (Fig. 10).The expression of VdWOX3, VdWOX7 and VdWOX9 was down-regulated.However, the expression of VdWOX4, VdWOX8, VdWOX11 and VdWOX12 was up-regulated.The expression of VdWOX2 was first down-regulated and then up-regulated.Moreover, the expression of VdWOX1, VdWOX5, VdWOX6 and VdWOX10 was first up-regulated and then down-regulated.

Discussion
The WOX gene family is one of the highly conserved transcription factor family in plants.Their function is essential for normal plant development and mutations often lead to severe defects.Due to their functional importance, current research on plants is both intensive and extensive.However, to date, studies on the function of VdWOX genes were very limited, and the members of the VdWOX gene family have not been systematically identified and defined.In this study, we identified 12 different VdWOX genes from the blueberry reference genome, which were distributed on eight blueberry chromosomes, and performed bioinformatic analyses of the gene family, including gene structure, conserved motifs, cis-regulatory elements, physicochemical properties of proteins, predicted structures and subcellular localization, which provided a framework for further studies of the gene family.
In this study and reported horticultural crops, the WOX family had been stably divided into three clades (WUS clade, intermediate clade and ancient clade).The two typical conservative domains of the WOX family were HD and WUS-box.In a previous study, the WUSbox domain existed in the WUS clade [3], which was once again confirmed in conservative motif analysis.From the evolutionary relationship of WOX family proteins, the WUS clade was the clade with the highest number of WOX family members, reaching 75%.Each gene in the blueberry WOX family had a synchronous corresponding gene in Arabidopsis and grape, indicating that the differentiation of the WOX family preceded the differentiation of the grape, blueberry and Arabidopsis species.In terms of evolutionary relationships, grape as a woody plant was more closely related to blueberry.The number of WOX family members in Arabidopsis, barley, grape and rice varied on different evolutionary branches, indicating that the WOX branches of each species underwent different evolution after species differentiation (Fig. 3) [23].
As a transcription factor, WOX contains a DNA-binding domain, a trans-regulatory domain, a nuclear localization signal and an oligomerisation site.The DNA-binding domain is the homologous domain of WOXs proteins, and previous studies had shown that AtWUS1, AtWOX3, AtWOX4 and AtWOX11 were localized in the nucleus in Arabidopsis thaliana [47].Moreover, PtoWUSa, Pto-WOX4a, PtoWOX5a, PtoWOX11/12a and PtoWOX13 were also localized in the nucleus in Populus [48].Through subcellular localization prediction, all of the VdWOX proteins were localized in the nucleus in blueberry (Table 1).
The gene structure and motif analysis results indicate that the WOX gene family in blueberries has a unique motif, and all genes contain motif 1 and motif 2. Different branches also have their own unique themes, for example, motif 6 only exists in the Intermediate Clade subfamily, while motif 6 only exists in the WUS Clade.Similarly, in Melastoma dodecandrum, AncientClade has motif 3, motif 6 and motif 10 that do not exist in other evolutionary branches.Intermediate Clade has a unique motif 4 [49].This phenomenon indicates that WOX genes in different evolutionary branches may have different functions.
The WOX gene family plays an important role in regulating plant growth and development, stress resistance, and plant hormone signal transduction [6,38].The promoter of VdWOX contains cis acting elements related to plant growth and development, hormones, and stress resistance.Research has shown that the WOX genes is regulated by hormones such as CK, IAA and ABA in the regulation of plant growth and development [16,50].In Arabidopsis, WUS exhibits a resistive effect and limits the IAA signaling pathway to maintain stem cell characteristics [51].The CK signal can directly activate the dynamic pattern of WUS expression, and the positive feedback loop between cytokinin signal and WUS function affects the stem meristem tissue pattern [52].Many hormone responsive elements were also observed in the VdWOX promoter region, including ABRE elements, auxin responsive elements, TGA elements, TCA elements, and GARE motifs.Therefore, the VdWOX gene family plays a crucial role in plant growth, development, and stress resistance in blueberries by mediating plant hormone regulation.
In Arabidopsis, AtWOX1 and AtWOX3 play a role in leaf development, especially leaf extension and margin development, and the phenotype of Arabidopsis single or double mutants exhibits narrow leaves [53].In leaves, VdWOX2 (the closest relative to AtWOX1) and VdWOX10 (the closest relative to AtWOX3) exhibited the highest expression levels (Fig. 8).This finding indicated a potential functional redundancy between these 2 genes in leaf development.AtWOX4 plays a crucial role in facilitating the growth of stem cells in both the procambium and cambium.It is specifically expressed in these two regions [53].Similarly, VdWOX3 and VdWOX10, which shares similarities with AtWOX4, also shows significant expression in the stem.AtWOX5 in Arabidopsis leads to ectopic flower formation and altered root morphology [54].Meanwhile, PtoWOX5a is involved in adventitious root development in poplar [55].VdWOX9 is closest to AtWOX5, which exhibited the highest expression level in roots, indicating that it functions in roots (Figs. 3 and 8).
Plants grow in a fixed location, but their surrounding environment is perennially changing, so they need to regulate their metabolism to adapt to environmental changes in order to meet their growth needs.Previous studies on WOX genes had focused on plant development, and few studies had been conducted on the stress caused by environmental changes.Our results indicated that VdWOX genes were indeed responsive to environmental stresses.Salt stress induced the expression of VdWOX genes.The expression of VdWOX2 and VdWOX10 reached a maximum at 9 days after 200mM NaCl treatment (Fig. 9).Additionally, the expression of VdWOX2 and VdWOX10 was found to be highest in leaves (Fig. 8).These findings suggested that blueberries might adapt to high salt environments by altering physiological and biochemical mechanisms of VdWOX regulation in leaves.Whereas drought treatment may repress VdWOX7 expression (Fig. 10).In the phylogenetic tree, VdWOX7 is the most closely related to Arabidopsis AtWUS1 (Fig. 3).VdWOX7 is primarily expressed in terminal buds and leaves (Fig. 8).However, during drought stress, the terminal buds and leaves are susceptible to damage, leading to a reduction in the expression of VdWOX7.
In recent years, WOX family members had been successively cloned and characterized in most plants, and the biological functions of some of them had been analyzed.The involvement of WOX in the regulation of plants roots, stems, leaves, flowers, fruits, seeds, embryonic development and abiotic stress had been reported in numerous studies [3,22,[56][57][58][59][60], which had led to a deeper understanding and appreciation of the role of WOX in the regulation of plant growth and development.These studies had led to a deeper knowledge and understanding of the role of WOX in the regulation of plant growth and development.However, studies on the involvement of WOX in the regulation of plant stress tolerance were still very limited, with relevant studies mainly focusing on plant species such as Arabidopsis thaliana [18], rice [43], poplar [44], cotton [61] and little relevant studies on horticultural crops.We used bioinformatics to identify the members of the VdWOX gene family, analyzed their gene structures, protein properties, tissue expression and the expression under stress in blueberry.It provides theoretical significance for the future in-depth analysis of the molecular mechanism of the role of VdWOX gene family.However, at the same time, our research also has limitations.The focus of our research is to use bioinformatics to identify the members of the WOX gene family in blueberries and predict their functions.Further research is needed to clarify the characterization of gene function and explain the mechanism of gene functional expression.At the same time, the excavation of VdWOX upstream regulators provides theoretical basis and new ideas for downstream target genes to regulate the plant growth and development processes and response to stress.
Blueberry seedlings were treated with 200 mM NaCl for 0 d, 1 d, 3 d, 5 d, 7 d and 9 d, and were treated with drought for 1 d, 3 d, 6 d, 9 d, 12 d and 15 d.After treatment, the tissues of the plants were taken and total RNA was extracted.

Fig. 1
Fig. 1 Chromosome distribution of WOX gene family members in blueberry.The left axis displays the length of each chromosome, estimated in megabases (Mb)

Fig. 2
Fig. 2 Sequence ratios of blueberry WOX gene family proteins for conserved structural domain analysis.(A) Phylogenetic tree analysis of WOX proteins in blueberry, I: WUS Clade, II: Ancient Clade, III: Intermediate Clade.(B) Domains characterised, light purple represents homeodomain, light blue represents Wus-box, sequence size (bp) is indicated by the scale at the bottom of the figure.(C) Sequence of VdWOX proteins homeodomain.(D) WUS-box sequence of VdWOX gene family proteins.(E) Secondary structure statistics of VdWOX proteins sequence

Fig. 4 Fig. 6 Fig. 5
Fig. 4 Conservative motif and gene structure analysis of VdWOXs.(A) The phylogenetic tree of VdWOXs, I: WUS Clade; II: Ancient Clade; III: Intermediate Clade.(B) CDS and intron structures, CDS and introns are represented by light green boxes and black lines, respectively.The sizes of their sequences (bp) are shown with a scale at the bottom of the figure.(C) Identification of the conserved motifs in VdWOXs.Boxes in different colors represent different motifs.The sizes of their sequences (aa) are shown with a scale at the bottom of the figure.(D) VdWOX gene family motif sequences

Fig. 7 Fig. 8 Fig. 9
Fig. 7 Analysis of Cis-regulatory element in the promoter of VdWOX genes.(A) Cis-regulatory element; (B) Statistics of Cis-regulatory element contained in VdWOX family genes

Fig. 10
Fig. 10 Expression pattern analysis of VdWOX genes under drought stress.After 1 d, 3 d, 6 d, 9 d, 12 d and 15 d of drought treatment, the transcription level of VdWOXs at 1 d was set to "1".Average values and standard deviation of three biological replicates were showed.Data are presented as the means ± SDs. biological replicates were used for the statistical analyses.Asterisks indicate significant differences (t test) compared to.*, **and*** indicate p-values < 0.05, < 0.01and < 0.001, respectively

Table 1
Physical and chemical properties of WOX gene and its encoding protein The results indicated that members of the WOX gene family were divided into WUS Clade, Ancient Clade, and Intermediate Clade, and the number of members of the WUS Clade was larger than that of the Ancient Clade and the Intermediate Clade.There were 44 members in the WUS branch, including 8 in Arabidopsis, 13 in maize, 6 in grape, 8 in barley and 9 in blueberry.There were 15 members in the ancient branch, including 3 in Arabidopsis, 6 in maize, 3 in grape, 2 in barley and 1 in blueberry.And there were 23 members in the Intermediate branch, including 4 in Arabidopsis, 11 in maize, 2 in grape, 4 in barley, and 2 in blueberry.The number of WOX family members varied greatly among different species in different evolutionary branches, suggesting that each species underwent different evolutions after differentiation, and the number of genes changed with the evolution of the gene family.